1. Field of the Invention
This invention relates to the staining of microorganisms. More particularly, it relates to the determination of the Gram sign by staining the microorganisms with a fluorescent dye or dye combination.
2. Description of the Prior Art
The Gram stain is one of the most widely used and important stains in microbiology. It permits the differentiation of microorganisms into two groups, conventionally referred to as Gram-positive and Gram-negative, based on the color of the stained organism at the conclusion of the staining protocol. In the Gram stain procedure, microorganisms on a cover slide are contacted with aqueous crystal violet, treated with iodine-potassium iodide solution, decolorized with alcohol or an ether-acetone mixture, counterstained with safranin O, dried, and examined. Gram-positive microorganisms retain the crystal violet and are thereby stained blue-violet. The Gram-negative microorganisms are completely decolorized by the organic solvent and show only the red color of the subsequently applied counterstain.
The determination of the Gram sign of a microorganism by the original procedure or any of the many subsequently described modifications is not a routine operation. In order to ensure accurate results, training, skill, proper technique and attention to detail are required. As a consequence, alternative procedures for Gram sign determination have been sought.
U.S. Pat. No. 4,225,669 to Melnick discloses a composition for staining both Gram-negative and Gram-positive microorganisms comprising a chelating agent and a non-fluorescent dye operative at a basic or neutral pH. Determination of the Gram sign may be carried out subsequent to staining by an acid wash which decolorizes the Gram-positive microogranisms.
Attempts to adapt staining with fluorescent dyes to Gram sign determinations have been disclosed. Staining of microorganisms with fluorescent dyes is well known and Gram-negative and Gram-positive microorganisms show different taining characteristics with fluorescent dyes. Govoronov et al., Microbiology 51, 587 (1982) reported that ethidium bromide (EB) does not stain intact E. coli cells because the dye does not penetrate the cell membranes, but that staining does occur if carried out in the presence of ethylenediamine tetraacetic acid, EDTA. Matsuyama, Microbiology Letters 21, 153 (1984), observed that rhodamine 123 stained twelve Gram-positive bacteria, but seven of fourteen Gram-negative stains were sparsely stained. One of the Gram-negative strains, Salmonella minnesota, stained only after treatment with EDTA.
U.S. Pat. No. 4,094,745 to Scholefield teaches a method for staining microorganisms in food samples with a fluorochrome in the presence of phosphate ions. A protocol to detect non-viable Gram-positive and Gram-negative microorganisms in a milk sample and to determine the Gram sign includes heating the microorganims in the presence of hydrochloric acid.
U.S. Pat. No. 4,126,516 to Messing et al. discloses a growth-based method for determination of the Gram sign of an unknown microorganism. The organism is cultured in/on a growth medium containing a lipophilic fluorescent material. During growth, Gram-negative microorganisms, having a higher cell membrane lipid content, incorporate more of the lipophilic dye than Gram-positive microorganisms which have a lower cell membrane lipid content. The Gram sign of the unknown is assigned by analysis of its fluorescence emission as compared with the fluorescence emission from known Gram negative and Gram-positive controls cultured under identical conditions.